Methods and compositions for assessing immune response in murine tumor models

Abstract

To provide methods for assessing immune response in murine models.SOLUTION: The present invention provides a method comprising: obtaining a sample from a murine model; detecting the expression level of a panel of genes in the sample, wherein the panel comprises at least 50 genes; comparing the expression level of the panel to a reference expression level; and assessing the immune response in the murine model.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a co-pending application of PCT / CN2020 / 094595 filed on June 5, 2020. No. 6,313,563, filed on May 13, 2003, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates generally to molecular biology, cancer biology and animal models. [Background technology]

[0003] Cancer is a heterogeneous disease and is highly related to the immune system. The importance of fully understanding the interactions between cancer and immunity in cancer treatment has been widely recognized. However, only 20-30% of cancer patients respond to immunotherapy and have immune-related There is also the added complication of inducing adverse effects and autoimmune responses. Who can benefit from immunotherapy? Being able to predict the outcome could save treatment costs and patient health. cormorant.

[0004] Experimental mouse models derived from a variety of biological techniques are essential preclinical model systems. Molecular pathology techniques, such as gene expression biomarkers, are enabling these models to be systemically profiled. However, to date, For mouse preclinical cancer immunotherapy studies to characterize tumor-immune interactions at the expression level There are currently no robust, cost-effective assays designed for this purpose. Summary of the Invention

[0005] In one aspect, the present disclosure provides a method for evaluating immune responses in a mouse model. In one embodiment, the method includes obtaining a sample from a mouse model, detecting a gene in the sample, The aim of the present invention is to detect the expression level of a panel of genes, the panel being selected from the group disclosed herein. The panel includes at least 50 selected genes, and the expression levels of the panel are compared to a reference expression level. This includes comparing the efficacy and safety of immunized mice with those of control mice and evaluating immune responses in mouse models.

[0006] In certain embodiments, the mouse model has a tumor. In certain embodiments, the sample It is tumor tissue.

[0007] In certain embodiments, the mouse model is treated with a therapy. , the therapy is immunotherapy.

[0008] In certain embodiments, expression of the panel of genes is determined using next generation sequencing (NGS). It is detected using

[0009] In certain embodiments, the immune response is mediated by B cells, dendritic cells, macrophages, monocytes, nascent cells, and the like. an immune cell selected from the group consisting of a CD4+ T cell, a CD8+ T cell, and a CD4+ T cell; This includes the activation of immune cells.

[0010] In another aspect, the present disclosure provides a kit for assessing immune responses in a mouse model. In certain embodiments, the kit provides for the detection of genes in a sample from a mouse model. The panel includes primers for detecting expression of a panel of antibodies selected from the group disclosed herein. The gene includes at least 50 genes selected from the set of

[0011] In another aspect, the present disclosure provides a method for evaluating immune responses in a mouse model using microarrays. In certain embodiments, the microarray provides a method for detecting chromatin patterns in a sample from a mouse model. The panel comprises probes for detecting the expression level of a panel of genes in the The present invention includes at least 50 genes selected from the group disclosed herein.

[0012] In yet another aspect, the present disclosure provides a method and apparatus for performing a method of a computer-readable medium having instructions stored thereon. When the instructions are executed by a processor, the instructions cause the processor to The expression level of a panel of genes in a sample, the panel being as disclosed herein. A search for at least 50 genes selected from the group of genes selected from the panel of expression levels; and evaluation of immune responses in mouse models. A computer readable medium is provided.

[0013] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure is incorporated herein by reference in its entirety in conjunction with the detailed description of specific embodiments presented herein. This can be better understood by reference to one or more of these drawings. [Brief description of the drawings]

[0014] [Figure 1] 1A and 1B show the results of cell sorting for B cells (FIG. 1A) and T cells (FIG. 1B) from non-tumor-bearing mouse spleens from a validation panel. [Diagram 2] Figure 2 shows quality assessment by comparing mouse cancer immunotherapy panel target and non-target gene expression levels in the validation panel. [Diagram 3] FIG. 3 shows single sample gene set enrichment analysis (ssGSEA) for 50 categorized mouse cancer immunotherapy panel signatures showing high consistency biomarkers in the validation panel. [Figure 4]FIG. 4 shows the estimated cell fractions in the validation panel using signatures based on mouse cross-cancer gene expression panels. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Before describing the present disclosure in more detail, it is to be understood that the disclosure is not limited to particular embodiments described. It should be understood that this may of course vary. are for purposes of illustrating particular embodiments only and are not meant to be limiting, Because the scope of the present disclosure is limited only by the appended claims. This should also be understood.

[0016] Unless otherwise defined, all technical and scientific terms used herein are intended to be understood as meaning those to which this disclosure pertains. The terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure. Although any method and material can be used, the preferred methods and materials are described herein.

[0017] All publications and patents cited herein are hereby incorporated by reference in their entirety. and are hereby incorporated by reference as if individually indicated to be incorporated by reference. Discloses methods and / or materials in connection with which the publications are incorporated and cited. All publications cited herein are incorporated by reference for the purposes of their disclosure. since its disclosure is prior to the filing date of the present application, and the present disclosure is hereby expressly incorporated by reference in its entirety. Nothing contained herein should be construed as an admission that no prior right exists. The dates of publication may be different from the actual publication dates which may need to be independently confirmed.

[0018] As will be apparent to those of skill in the art upon reading this disclosure, the particular embodiments described and illustrated herein may Each of the above may be implemented in several other embodiments without departing from the scope or spirit of the present disclosure. A separate component that can be easily separated from or combined with any of the characteristics of the state Any recited method must conform to the recited order or logical order of events. Any other possible order may be performed.

[0019] definition

[0020] The following definitions are provided to assist the reader. Unless otherwise specified, as used herein All technical terms, notations and other scientific or medical terms or technical terms used herein are the property of their respective owners. The terms used herein shall have the meaning commonly understood by those of ordinary skill in the science and medical arts. In this regard, terms having their commonly understood meanings have been omitted for clarity and / or ease of reference. As defined herein by reference, and incorporation of such definitions into this specification is not intended to be limiting. , to represent substantial differences in the definition of terms as commonly understood in the industry. should not necessarily be interpreted as

[0021] As used herein, the singular forms "a," "an," and "the" are used where the context clearly indicates. Plural references are included unless otherwise indicated.

[0022] The terms "amount" or "level" refer to the amount of a polynucleotide of interest or a target molecule present in a sample. Such amount refers to the amount of a polypeptide in an absolute sense, i.e., the amount of a polypeptide in a sample. Either expressed as a total amount of nucleotide or polypeptide, or relatively, i.e. , may be expressed as the concentration of polynucleotide or polypeptide in the sample.

[0023] As used herein, the term "cancer" or "tumor" refers to a tumor that is caused by abnormal cell proliferation. Any disease means all stages and stages of a disease affecting any tissue, organ or cell in the body. The term includes all forms of tumors characterized as malignant, benign, soft tissue or solid. All known cancers and neoplastic conditions, as well as all stages of cancer, including pre- and post-metastatic cancers In general, cancer is classified according to the location of the cancer or the type of cancer that develops. They can be classified according to the tissue or organ in which they originate and the morphology of the cancerous tissues and cells. As used herein, types of cancer include, but are not limited to, acute lymphoblastic leukemia, Leukemia (ALL), acute myeloid leukemia, adrenal cortical carcinoma, anal cancer, astrocytoma, pediatric cerebellar or basal cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, cerebellar astrocytoma, cerebral astrocytoma Cell tumors / malignant gliomas, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and Hypothalamic glioma, breast cancer, Burkitt's lymphoma, cervical cancer, chronic lymphocytic leukemia, Myeloid leukemia, colon cancer, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma , retinoblastoma, gastric (stomach) cancer, glioma, head and neck cancer , heart cancer, Hodgkin's lymphoma, pancreatic islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), pharyngeal cancer, leukemia, liver cancer, lung cancer, neuroblastoma, non-Hodgkin's lymphoma , ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retina Blastocytoma, Ewing family tumors, skin cancer, stomach cancer, testicular cancer, pharyngeal cancer, thyroid cancer These include adenocarcinoma and vaginal cancer.

[0024] As used herein, a "cell" may be a prokaryotic or eukaryotic cell. Eukaryotic cells include, for example, bacteria. Eukaryotic cells include, for example, fungi, plant cells and animal cells. Types of animal cells (e.g., mammalian or human cells) include, for example, circulating Cells of the immune system or organs (e.g., B cells, T cells (cytotoxic T cells, natural Killer T cells, regulatory T cells, helper T cells), natural killer cells, granulocytes (e.g. basophilic granulocytes, eosinophilic granulocytes, neutrophilic granulocytes and hyperlobulated neutrophils), mononuclear cells or macrophages, red blood cells (e.g., reticulocytes), mast cells, platelets or megakaryocytes, and dendritic cells), cells of the endocrine system or organs (e.g., thyroid cells (e.g., thyroid gland parathyroid cells (e.g., parathyroid chief cells, eosinophilic cells), adrenal cells cells of the pineal gland (e.g., pinealocytes), nervous system or cells of an organ (e.g., glioblastoma cells (e.g., astrocytes and oligodendrocytes), Microglia, magnocellular neurosecretory cells, astrocytes, Bechtel cells and pituitary cells (e.g. , gonadotrope cells, adrenocorticotrope cells, thyrotrope cells cells, somatotrophs and lactotrophs), respiratory system or organs Cells (e.g., alveolar cells (type I pneumocytes and type II pneumocytes), Clara cells, goblet cells , alveolar macrophages), cells of the circulatory system or organs (e.g., cardiac myocytes and peripheral cells) , cells of the digestive system or organs (e.g., gastric chief cells, parietal cells, goblet cells, Paneth cells, Gastrointestinal Cells, D cells, ECL cells, I cells, K cells, S cells, enteroendocrine cells, enterochromaffin cells , APUD cells, cells of the liver (e.g., hepatocytes and Kupffer cells), envelope system or They stimulate the cells of organs, such as bone cells (e.g. osteoblasts, osteocytes and osteoclasts), dental cells, and cells of the nucleus (e.g., cementoblasts and ameloblasts), cells of the cartilage (e.g., chondroblasts and chondrocytes), skin / hair cells (e.g., hair follicles, keratinocytes and melanocytes ( Nevus cells), muscle cells (e.g., myocytes), adipocytes, fibroblasts and tendon cells), secretory cells Cells of the urinary system or organs (e.g., podocytes, juxtaglomerular cells, intraglomerular mesangial cells) , extraglomerular mesangial cells, renal proximal tubule brush border cells and macula densa cells) and Cells of the reproductive system or organs (e.g. sperm, Sertoli cells, Leydig cells, eggs, oocytes) Cells may be normal, healthy cells or diseased, unhealthy cells (e.g. The cells may further include mammalian zygotic or embryonic stem cells, fetal cells, and the like. Stem cells include embryonic stem cells, induced pluripotent stem cells and adult stem cells. They can differentiate into specialized cell types through cycles of cell division while maintaining their differentiated state. Stem cells are classified into totipotent stem cells, pluripotent stem cells, multipotent stem cells, and oligopotent stem cells. Stem cells may be either unipotent or unipotent stem cells, both of which can be derived from somatic cells. The cells can also include cancer stem cells. Mammalian cells can be rodent cells, e.g., mouse cells. Mammalian cells may be mouse, rat or hamster cells. Mammalian cells may be lagomorph cells, e.g., mouse, rat or hamster cells. The mammalian cell may also be a primate cell, e.g., a human cell. In certain instances, the cells may be cells used in mass bioproduction, e.g. , CHO cells.

[0025] The term "complementarity" refers to the ability of a molecule to be complementary to another molecule, whether in the traditional Watson-Crick or other non-traditional manner. By either one or the other, we mean the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence. Complementarity can occur by forming hydrogen bonds (e.g., Watson-Crick base pairs) with a second nucleic acid sequence. The percentage of residues in a nucleic acid molecule that can be sequenced (e.g., 5, 6, or 10) 7, 8, 9, 10 are 50%, 60%>, 70%>, 80%>, 90% and 100% complementary "Perfectly complementary" means that all contiguous residues of a nucleic acid sequence are identical to those of a second nucleic acid sequence. As used herein, "substantially" means "to hydrogen bond with the same number of consecutive residues in a row." "Complementary to" is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, and or more nucleotides over a region of at least 60%, 65%, 70%, or 75% , 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% complementary It indicates the degree of sexual compatibility or the number of nuclei that hybridize under stringent conditions. It means acid.

[0026] In this disclosure, the terms "comprises," "comprised," and "comprising" are used interchangeably. Terms such as "rising," "contains," and "containing" are , have the meaning given to them in U.S. patent law, are generic or open ended, and may include additional Note that the disclosure does not exclude any unrecited elements or method steps. "consisting essentially of" and "consist essentially of" Terms such as "essentially of" have the meaning ascribed to them in the United States Patent Act and are intended to be used in the context of the claimed subject matter. Contains additional components or steps that do not materially affect the basic and novel characteristics of the invention The terms "consists of" and "consist "G of" has the meaning ascribed to it in U.S. patent law, i.e., these terms are patent This is Rose End.

[0027] The terms "determine," "assess," "assay," "measure," and "detect" " can be used interchangeably and refer to both quantitative and semi-quantitative determinations. When referring to either quantitative or semi-quantitative determination of a polynucleotide or polypeptide of interest, "Determining the level" of a polypeptide or "detecting" a polynucleotide or polypeptide of interest. The phrase "to be" may be used.

[0028] The term "hybridizing" refers to the binding of a specific nucleotide sequence under stringent conditions. The term refers to the preferential binding, duplex formation or hybridization of nucleic acid molecules to a target nucleic acid. "Stringent conditions" are used when testing a mixed population (e.g., cell lysates or a probe hybridizes preferentially to its target subsequence in the DNA preparation; It hybridizes to a relatively small extent or not at all to other sequences. In the case of nucleic acid hybridization (e.g., (such as in rastering, microarray, Southern or Northern hybridization) "Stringent hybridization" and "stringent hybridization" "Solution wash conditions" are sequence dependent and vary under different environmental parameters. Extensive guidelines for hybridization are available, for example, from the Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I,Ch.2,“Overview of principal les of Hybridization and the strategy of Nucleic acid probe assays,”(1993)Elsevi er, NY. Generally, high stringency hybridization and washing conditions are determined by the melting temperature (Tm) of a specific sequence at a defined ionic strength and pH. The Tm is chosen to be approximately 5° C. lower than the target (at defined ionic strength and pH). This is the temperature at which 50% of the sequence hybridizes to a perfectly matched probe. The sensitive conditions are chosen to be equal to the Tm of the particular probe. has more than 100 complementary residues on the array or filter in Northern blots. Stringent hybridization conditions for hybridization of complementary nucleic acids An example of hybridization conditions is 42°C using a standard hybridization solution (e.g., Sambrook and Russell Molecular Cloning :A Laboratory Manual(3rd ed.)Vol.1-3(200 1)Cold Spring Harbor Laboratory,Cold Spr. (See E. G. Hartman, NY, School of Pharmaceutical Sciences, Boston, MA). High stringency washing conditions An example of stringent washing conditions is 0.15M NaCl at 72° C. for about 15 minutes. An example of the conditions is a 0.2×SSC wash at 65° C. for about 15 minutes. A low stringency wash was performed prior to the high stringency wash to remove the end probe signal. For example, a moderate stringency wash for duplexes longer than 100 nucleotides is used. An example of a reagent wash is 1×SSC at 45° C. for 15 minutes. An example of a low stringency wash for a duplex over an octide is 4x SSC to 6x SSC. SC at 40°C for 15 minutes.

[0029] The term "mouse model," as used herein, refers to a mouse model for the development of a disease or disorder, such as cancer. and progression, as well as to test treatments before giving them to humans. It means mouse.

[0030] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to deoxyribonucleic acid. polymeric forms of nucleotides of any length, either mono- or ribonucleotides, or Polynucleotides can have any three-dimensional structure and their analogs. The polynucleotide may serve any function, known or unknown. Typical examples include genes, gene fragments, exons, introns, messenger RNA (mRNA), and RNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, shRN A, single-stranded short or long RNA, recombinant polynucleotide, branched polynucleotide Tides, plasmids, vectors, isolated DNA of any sequence, regulatory regions, The nucleic acid molecules include isolated RNA, nucleic acid probes and primers. may be cyclic.

[0031] The term "oligonucleotide" refers to a nucleic acid having at least about 5 nucleotides and up to about 500 nucleotides. (For example, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 21, 22, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 , 125, 150, 175, 200, 250, 300, 350, 400, 450 or 5 In some embodiments, for example, an oligonucleotide sequence is used. The nucleotide may be from about 15 nucleotides to about 30 nucleotides, or from about 20 nucleotides to about 2 5 nucleotides, which can be used, for example, in polymerase chain reaction (PCR) amplification as primers in ELISAs and / or hybridization assays or The oligonucleotides of the present invention may be used as primers in microarrays. The peptides may be natural or synthetic, as is well known in the art, e.g., DNA, RNA, A, PNA, LNA, modified backbones, etc.

[0032] The term "sample" or "biological sample" as used herein refers to one or more Any cell, tissue, organoid, or any other sample that contains a nucleic acid molecule of interest. In certain embodiments, the sample may comprise a cell (e.g., a normal cell, a cancer cell, a cell line), a tissue, or a combination of both. tissues (e.g., normal tissues, cancer tissues, xenograft or allograft tissues), organoids, etc. be.

[0033] The term "substrate," when used in reference to an array, refers to a substrate that holds the associated assay components (e.g., The term "substrate" refers to a material capable of supporting an assay region, a cell, a test compound, etc. Examples include, but are not limited to, glass, Si-based materials, functionalized polystyrene, Polyethylene glycol, functionalized organic polymers, nitrocellulose or naphthalene. Suitable substrates include iron film, paper, cotton, and synthetic materials. The substrate does not need to be flat. The material bound to the substrate may be in any type of shape, including spherical (e.g., beads). It may be attached to any part of the substrate (e.g., attached to the interior of the porous substrate material). A preferred embodiment of the present technology has a nucleic acid probe attached to a substrate. A lobe is a group that is associated with a substrate by non-random chemical or physical interactions. In some preferred embodiments, the attachment is provided, for example, by a linker. By covalent bonding, as in

[0034] The term "tumor sample" refers to a biological sample or organism that contains one or more tumor cells. Biological samples include samples from any source. Biological samples include body fluids, such as blood, plasma, serum or A urine sample or, for example, by biopsy, a cell, tissue or organ, preferably a cancer cell, Obtained from tumor tissue suspected of containing cells or consisting essentially of cancer cells Samples are included.

[0035] Gene Panels for Assessing Immune Responses

[0036] Malignant tumors do not only contain cancer cells; normal cells from the body also infiltrate the tumor. For example, various immune cells that infiltrate tumors help detect and kill cancer cells. This has important implications for tumor progression and response to therapy. Although these signals are often studied in tumors, different cells, such as immune cells, may also play a role in tumor function. The ratio of sialyl species has rarely been studied at the gene expression level.

[0037] The methods and compositions described herein are directed, in part, to the differentiation and proliferation of various immune cells in tumors. The present invention is based on the discovery of a group of genes that can be used to identify and evaluate immune responses, such as immune ratios. In certain embodiments, the tumors are obtained in a mouse tumor model. In this study, genes were selected based on genomic data from non-tumor-bearing and tumor-bearing mice. In certain embodiments, a group of genes is used to identify B cells, dendritic cells, macrophages, and the like. A small number of cells, including phages, monocytes, natural killer cells, CD4+ T cells and CD8+ T cells. At least eight different immune cell types can be identified. The subgroup of patients will focus on key pathways at the tumor interface: the immune system, the tumor microenvironment, and the immune response. Surface and transcriptome biomarkers and data normalization The gene includes an internal reference gene for amplification.

[0038] In certain embodiments, the group of genes used herein is selected from the group shown in Table 1. will be done.

[0039] Methods for detecting expression levels of gene panels

[0040] In one aspect, the present disclosure provides a method for evaluating immune responses in a mouse model. In certain embodiments, the method includes obtaining a sample from a mouse model, detecting in the sample and detecting the expression level of a panel of genes in the subject, the panel being selected from the group shown in Table 1. At least 50, 100, 150, 200, 250, 300, 350, 4 00, 450, 500, 550, 600, 650, 700, 750, 800, 850, 9 Contains 00, 950, and 1000 genes, and compares the expression levels of the panel to a reference expression level and evaluating the immune response in a mouse model.

[0041] In certain embodiments, the expression levels of the gene panel include, but are not limited to, increased including breadth assays, hybridization assays and sequencing assays, Detection may also be at the RNA (e.g., mRNA) level using suitable methods known in the art. good.

[0042] Amplification assays

[0043] Nucleic acid amplification assays involve copying a target nucleic acid (e.g., DNA or RNA) and then The amplification process includes an increase in the number of copies of an amplified nucleic acid sequence. Amplification can be exponential or linear. Examples of nucleic acid amplification methods include, but are not limited to, polymerase chain reaction (PCR) and polymerase chain reaction (PCR). PCR ("PCR", U.S. Patent Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide To Methods And App lications (Innis et al., eds, 1990), reverse transcriptase polymerase remerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR); TaqMan®, nested PCR, ligase chain reaction (Abravaya, K.,et al.,Nucleic Acids Research,23:675- 682, (1995)), branched DNA signal amplification (Urdea, MS See, et al., AIDS, 7(suppl 2):S11-S14, (1993). (Refer to references), amplifiable RNA reporters, and Q-β replication (Lizardi et al., Biotechnology (1988) 6:1197), based on transcription Amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA ( 1989)86:1173-1177), boomerang DNA amplification, strand displacement activation, cycle Ring probe technology, a self-sustaining sequence replication technique (Guatelli et al., Proc. Natl.Acad.Sci.USA(1990)87:1874-1878), Lori No. 5,854,033; Nucleic Acid Sequence-Based Amplification (NASB A) and quantitative PCR-based amplification such as serial analysis of gene expression (SAGE). Can be enjoyed.

[0044] In certain embodiments, the nucleic acid amplification assay is a PCR-based method. R begins with a pair of primers that hybridize to the target nucleic acid sequence to be amplified. The target nucleic acid sequence is then used as a template and dNTPs as building blocks to generate new The primer is extended by polymerase, which synthesizes a new strand and a target strand. The target strand is denatured to allow the primer to bind for the next cycle of extension and synthesis. After multiple amplification cycles, the total number of copies of the target nucleic acid sequence increases exponentially. If the nucleic acid obtained from the sample is RNA, the amplification step can optionally include This may include a reverse transcription step to generate cDNA of the RNA in The cDNA is amplified using primers to allow detection of the expression level of the gene of interest. to.

[0045] In certain embodiments, an intercalator that generates a signal when inserted into double-stranded DNA. Examples of agents include SYBR GREEN™ and S These agents include YBR GOLD™. These agents are not template specific and therefore do not The molecules are thought to be generated based on template-specific amplification. This is because the melting temperature of the template sequence is In general, the signal intensity is much higher than that of, for example, primer-dimers, so it depends on the temperature. This can be confirmed by monitoring the null.

[0046] In certain embodiments, a detectably labeled primer or a detectably labeled The probe is used to detect the mRNA of the gene of interest that corresponds to the primer or probe. (or cDNA reverse transcribed from mRNA). The method comprises the steps of: A probe can be used to allow simultaneous detection of the expression of multiple genes of interest.

[0047] Hybridization assay

[0048] Nucleic acid hybridization assays use probes to hybridize to target nucleic acids. Hybridization assays are used to generate a hybridization signal that allows detection of the target nucleic acid. Non-limiting examples include Northern blotting, Southern blotting, in situ hybridization, and Based on chromatin synthesis, microarray analysis, and multiplex hybridization The assays include:

[0049] In certain embodiments, the probes for the hybridization assay include detectable In certain embodiments, the nucleic acid is labeled for hybridization assays. The acid-based probes are unlabeled. Such unlabeled probes are suitable for use in microarrays. The nucleic acid molecule is then hybridized to a detectably labeled target nucleic acid molecule that can be immobilized on a solid support such as a plate. It can be bridging.

[0050] In certain embodiments, hybridization assays are performed using nucleic acids (e.g., RNA or to isolate nucleic acids (e.g., by gel electrophoresis) and The separated nucleic acids are then subjected to suitable incubation conditions in which the probes hybridize to the target nucleic acid and allow detection. The experiment was carried out by transferring the samples to a suitable membrane filter (e.g., nitrocellulose filter). For example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition on,Cold Spring Harbor Laboratory Press,1 See 989, Chapter 7. Hybridization of Probe and Target Nucleic Acids The reaction can be detected or measured by methods known in the art. For example, hybridization Autoradiographic detection of the hybridization was performed by photographing the hybridized filters. This can be done by exposing to a film.

[0051] In some embodiments, the hybridization assay is performed on a microarray. Microarrays provide a method for the simultaneous measurement of large numbers of target nucleic acid molecules. The nucleic acid may be RNA, DNA, cDNA reverse transcribed from mRNA, or chromosomal DNA. The target nucleic acid may be up to several million probes per square centimeter of substrate surface. A microarray comprising a substrate having a plurality of immobilized nucleic acid probes arranged at a density of 100 nm. The RNA or DNA in the sample can be allowed to hybridize to the The hybridization occurs when the DNA fragment is hybridized to a complementary probe, which is then detected by laser scanning. Hybridization intensities are determined for each probe on the array, and RNA or DNA The results are converted into a quantitative value that represents the relative level of A. Nos. 800,992 and 6,020,135, 6,033,860 and 6, See No. 344,316.

[0052] Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in U.S. Pat. No. 5,384,433. ,261. Although planar array surfaces are often used, the arrays are actually The array may be fabricated on any shape of surface, and may be fabricated on multiple surfaces. The material may be a bead, a gel, a polymer surface, a fiber such as an optical fiber, glass or any other suitable material. On other suitable substrates, which may be peptides or nucleic acids, see U.S. Pat. No. 5,770,355. No. 8, No. 5,789,162, No. 5,708,153, No. 6,040,193 and Arrays may be used in diagnostic procedures or other comprehensive devices. Useful microarrays may also be commercially available. For example, Nano String Technologies' Affymetrix x, QuantiGene 2.0 Multiplex Assay from Panomix y microarray.

[0053] Sequencing methods

[0054] Sequencing methods useful for measuring the expression of gene panels include sequencing of target nucleic acids. Any sequencing method known in the art can be used to identify the gene of interest. In general, sequencing methods include traditional and hybrid methods. It can be classified as high-throughput sequencing (next-generation sequencing). Sequencing methods include Maxam-Gilbert sequencing (chemical sequencing) These include the Sanger sequencing (also known as chain termination sequencing) and the Sanger sequencing (also known as chain termination sequencing). do.

[0055] High-throughput sequencing, or next-generation sequencing, is a method for sequencing a number of genes, such as the Sanger sequence. By using a methodology that is distinct from traditional methods, it is highly scalable and In this way, the entire genome or transcriptome can be sequenced at once. High-throughput sequencing includes sequencing by synthesis, sequencing by ligation, and sequencing, ultra-deep sequencing (Marguiles et al., As described in Nature 437(7057):376-80(2005) Sequencing by synthesis involves the use of labeled sequences in polymerase amplification. The complementary strand of the target nucleic acid is synthesized by incorporating a nucleotide or nucleotide analogue. Immediately following or upon successful incorporation of a labeled nucleotide, The signal of the label is measured and the identity of the nucleotide is recorded. The detectable label on the octide is removed prior to incorporation, and the detection and identification steps are repeated. Examples of sequencing by synthesis are known in the art, see, for example, U.S. Patent No. 7,333,636. ,056,676, U.S. Pat. No. 8,802,368 and U.S. Pat. No. 7,169,5 No. 60, the contents of which are incorporated herein by reference. Sequencing is performed using foldback PCR and anchored primers on a solid-phase substrate. The target nucleic acid fragments may be amplified by an amplicons. By hybridizing to the card primer, it can be bound to a solid phase surface and form a bridge amplified This technology can be used, for example, in Illumina® sequencing platforms. It is used in the form.

[0056] Pyrosequencing involves hybridizing a target nucleic acid region to a primer. , and deoxyribonuclease corresponding to the bases A, C, G, and T (U) in the presence of a polymerase. It involves the sequential incorporation of nucleotide triphosphates to extend the new strand. Each base incorporation is accompanied by the release of pyrophosphate, which is converted to ATP by sulfurylase. This drives the synthesis of oxyluciferin and the emission of visible light. The light emitted is equimolar to the number of nucleotides added in any one step. The number of hits is proportional to the number of hits. This process is repeated until the entire sequence is determined.

[0057] In certain embodiments, the expression of a gene of interest is measured using whole transcriptome shotgun analysis. It is detected by sequencing (RNA sequencing). The method of ion exchange has been described (Wang Z, Gerstein M and S nyder M, Nature Review Genetics (2009)10:5 7-63;Maher CA et al.,Nature(2009)458:97- 101;Kukurba K & Montgomery SB,Cold Sprin g Harbor Protocols(2015)2015(11):951-969 In certain embodiments, for example, oligonucleotide-based hybridization is used. Hybridization / capture techniques are used to pretreat samples undergoing RNA sequencing. In certain embodiments, the expression product of the gene of interest is enriched by hybridization. The probes used in the fusion / capture techniques are 20 to 200 nucleotides long, 500 nucleotides, 20-1000 nucleotides, 20-2000 nucleotides, 20 The length may range from 5000 nucleotides, or from 20 to 7000 nucleotides.

[0058] Comparison with reference levels

[0059] In certain embodiments, the methods disclosed herein include detecting expression levels of a panel of genes. The method includes comparing the signal to a reference expression level.

[0060] The term "reference expression level" refers to an expression level representative of a reference sample. In some embodiments, the reference sample is obtained from a healthy (e.g., non-tumor-bearing) subject or tissue. In some embodiments, the reference sample is a cancer or tumor tissue. The agent may be, for example, a tumor or a treatment (e.g., an anti-tumor therapy, e.g., anti-PD-1, anti-PD-L1, etc. The immune response was activated by the anti-immune checkpoint antibody In certain embodiments, the reference expression level is obtained from a subject or tissue in a test sample. The same or compatible measurement method as that used to detect the expression level of the target gene in The concentration of the ion exchange receptor is determined using a known method or assay.

[0061] In certain embodiments, the reference expression level may be predetermined. For example, the reference expression level The bell is a standard for collection of common cancer or tumor samples or tissues from the same tumor type. The expression level of a target gene may be calculated or generalized based on the measurement of the expression level of the target gene. Reference expression levels are those in average cancer or tumor samples from a general cancer or tumor population. The expression level of the target gene may be based on statistics generally accepted in the world.

[0062] In certain embodiments, the comparison step in the methods provided herein includes detecting determining the difference between the determined expression level and a reference expression level. The difference may be an increase or a decrease.

[0063] In certain embodiments, the difference from the reference expression level is further compared to a threshold value. In this embodiment, the threshold value is set so that if a difference from the reference expression level reaches the threshold value, such a difference A useful statistic can be established by statistical methods so that the results can be considered statistically significant. The quantitative analysis method is LDFisher & G.vanBelle,Biostati stics:A Methodology for the Health Science ces (Wiley-Interscience, NY, 1993). Statistical significance may be determined based on confidence ("p") values, using an unpaired two-tailed t-test. For example, it can be calculated as 0.1, 0.05, 0.025, or less than 0.01. P-values ​​can usually be used to indicate statistical significance. Confidence intervals and p-values ​​are well known in the art. This can be determined by well-known methods, e.g., Dowdy and Wearden, Sta. tistics for Research,John Wiley & Sons,N See, e.g., New York, 1983.

[0064] Evaluation of antitumor therapy

[0065] In another aspect, the present disclosure further provides a method for the detection of a cancer cell line comprising the steps of: In certain embodiments, the present invention provides a method for evaluating a treatment in a mouse model based on the The method includes administering a treatment to the mouse model, obtaining a sample from the mouse model, and and detecting the expression level of a panel of genes in the human ovarian tumor cell line, the panel being selected from the group consisting of those shown in Table 1. the expression level of the panel is reference expression level; These include comparing levels of IgG1 and evaluating immune responses in mouse models.

[0066] In certain embodiments, treatment includes administering a steroid to regulate or inhibit hormone action on the tumor. Antihormonal agents include natural or synthetic organic or peptidic compounds that act in a similar manner to hormone receptors.

[0067] In certain embodiments, the treatment includes a VEGFR inhibitor, an integrin receptor antagonist, These include angiogenesis inhibitors, including agonists and integrin antagonists.

[0068] In certain embodiments, the treatment includes tumor cell apoptosis promotion or apoptosis stimulation. Contains agents.

[0069] In certain embodiments, treatment includes the use of erbB2 receptor inhibitors, other protein tyrosine kinase inhibitors, Kinase inhibitors; ras inhibitors; raf inhibitors; MEK inhibitors; mTOR inhibitors; Cytochrome P450 inhibitors Clin-dependent kinase inhibitors; protein kinase C inhibitors; and PDK-1 inhibitors. Which include signal transduction inhibitors.

[0070] In certain embodiments, the treatment includes an anti-proliferative agent. Anti-proliferative agents include, for example, enzymes Inhibitors of farnesyl protein transferase and the receptor tyrosine kinase PD These include inhibitors of GFR.

[0071] In certain embodiments, the treatment includes administering an antibody, such as an antibody that specifically binds to an immune checkpoint. These include cancer immunotherapy agents. Immune checkpoints include, for example, A2AR, B7.1 , B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, CD48, CD160, CD244, CTLA-4, ICOS, LAG-3, LILRB1, LIL RB2, LILRB4, OX40, PD-1, PD-L1, PD-L2, SIRP-ALF (CD47), TIGIT, TIM-3, TIM-1, TIM-4, and VISTA Includes:

[0072] Kits and Microarrays

[0073] In another aspect, the disclosure provides a kit for use in the aforementioned methods. The kit comprises may contain any or all of the reagents for carrying out the methods described herein. In certain embodiments, the kit comprises a panel of genes in a sample from a mouse model. In certain embodiments, the panel comprises primers for detecting expression of the genes listed in Table 1. At least 50, 100, 150, 200, 250, 300 selected from the group shown , 350, 400, 500, 550, 600, 650, 700, 750, 800, 850 , 900, 950, 1000 genes.

[0074] In certain embodiments, the kit further comprises detecting the expression of a panel of genes using primers. The kit further comprises an agent for amplifying a product, e.g., mRNA. and instructional materials containing directions (i.e., protocols) for the practice of the provided methods. Instructional materials typically include, but are not limited to, written or printed materials. Such instructions may be stored and communicated by the end user. Any medium capable of carrying out the above-mentioned functions is contemplated by the present invention. Such mediums include, but are not limited to, However, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical Such media includes, but is not limited to, instructional media (e.g., CD-ROMs) such as instructional materials. A link to a website may include an address to an internet site that provides the service.

[0075] In another aspect, the present disclosure provides expression levels of a panel of genes in samples from a mouse model. In certain embodiments, oligonucleotide probes for detecting the marker are provided. The panel may comprise at least 50, 100, 150, 200, or more of the following antibodies selected from the group shown in Table 1: , 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 , 750, 800, 850, 900, 950, 1000 genes.

[0076] In certain embodiments, the probe is a nucleic acid sequence similar to that described in, e.g., Eds., Bowtell and S ambrook DNA Microarrays:A Molecular Clon ing Manual(2003)Cold Spring Harbor Labor A solid support such as an array slide or chip as described in Academic Press The structure of such devices is described in, for example, U.S. Pat. US Patent No. 5,837,832, PCT Application No. WO95 / 11995, U.S. Patent No. No. 807,522; U.S. Patent Nos. 7,157,229, 7,083,975, and 6, No. 444,175, No. 6,375,903, No. 6,315,958, No. 6,295, 153 and 5,143,854, No. 2007 / 0037274, No. 2007 / No. 0140906, No. 2004 / 0126757, No. 2004 / 0110212, No. 2004 / 0110211, 2003 / 0143550, 2003 / 00030 32 and 2002 / 0041420, are well known in the art. Nucleic acid arrays are also reviewed in the following references: Biotechnol Ann. u Rev(2002)8:85-101;Sosnowski et al.Psyc hiatr Genet(2002)12(4):181-92;Heller,Ann u Rev Biomed Eng(2002)4:129-53;Kolchinsk y et al.,Hum. Mutat(2002)19(4):343-60; and and McGail et al., Adv Biochem Eng Biotechno l(2002)77:21-42.

[0077] Microarrays are a set of, usually synthetic, antisense polynucleotides fixed to a solid support. It is composed of a large number of unique single-stranded polynucleotides, either fragments of DNA or cDNA. A typical polynucleotide is preferably about 6 to 60 nucleotides in length, and more preferably about 6 to 60 nucleotides in length. Preferably about 15 to 30 nucleotides in length, most preferably about 18 to 25 nucleotides in length. For certain types of arrays or other detection kits / systems, only about 7- It may be preferable to use oligonucleotides that are 20 nucleotides in length. Other types of arrays, such as those used in conjunction with chemiluminescence detection techniques, are preferred. The length of the probe is, for example, about 15 to 80 nucleotides, preferably about 50 to 70 nucleotides. The nucleic acid sequence is preferably about 55 to 65 nucleotides long, and more preferably about 60 nucleotides long. It may be a punch length.

[0078] Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in U.S. Pat. No. 5,384,433. ,261. Although planar array surfaces are often used, the arrays are actually The array may be fabricated on any shape of surface, and may be fabricated on multiple surfaces. The material may be a bead, a gel, a polymer surface, a fiber such as an optical fiber, glass or any other suitable material. Other suitable substrates may also include nucleic acids, see U.S. Pat. No. 5,770,358, U.S. Pat. Nos. 89,162, 5,708,153, 6,040,193 and 5,800 Arrays can be used to enable diagnostic or other operations on a global device. The food product may be packaged in such a way that

[0079] The probes and primers necessary to practice the present invention can be synthesized using well-known techniques. The oligonucleotides used as probes and primers can be synthesized or labeled. Nucleotides are described in Beaucage and Caruthers, Tetrahedron on Letts. (1981) 22:1859-1862 The solid-phase phosphoramidite triester method was used to synthesize the Needham-Van Devant er et al., Nucleic Acids Res. (1984)12:6159 It can be chemically synthesized using an automated synthesizer as described in US Pat. No. 6,168, 2003-010343.

[0080] COMPUTER-IMPLEMENTED METHODS, SYSTEMS AND APPARATUS - Patent application

[0081] Any of the methods described herein may be used in whole or in part to carry out the steps. 2. A computer system including one or more processors, the ... being configured to Thus, the embodiments may be implemented in accordance with each step or steps of the The methods described herein may have different components for carrying out each group. The present invention is directed to a computer system configured to perform any of the steps of Although presented as numbered steps, the steps of the methods herein may be performed simultaneously: In addition, some of these steps may be performed in different orders. It may be used with some of the other steps of the method, or with all or part of the steps. Any step of any method may be optional or partially optional. The present invention may be implemented in a module, circuit, or other means to perform the above-described functions.

[0082] Any of the computer systems referred to herein may include any suitable number of subsystems. In some embodiments, the computer system may include a subsystem Other implementations include a single computing device, of which the components may be components. In this form, a computer system has internal components, each of which is a subsystem. The subsystems are connected via a system bus. Further subsystems can be interconnected, e.g. printers, keyboards, etc. , storage devices, monitors connected to display adapters, and more. The peripherals and input / output (I / O) devices that are connected to the controller are The device may be linked to a computer system by any number of means known in the art. For example, a serial port or an external interface (e.g., Ethernet , Wi-Fi) is a method of connecting computer systems to wide area networks such as the Internet. It can be used to connect to a mouse input device or a scanner. Interconnection via the subsystems allows the central processing unit to communicate with each subsystem and to A pointer from a storage device (e.g. a fixed disk such as a hard drive or optical disk) It allows control of the execution of the program and allows information exchange between subsystems. The memory and / or storage device may incorporate a computer readable medium. Any of the data mentioned in the document may be output from one component to another, It may also be output to a user.

[0083] The computer system may, for example, be connected to an external interface or to an internal interface. It may contain multiple identical components or subsystems linked together by interfaces. In some embodiments, the computer system, subsystem, or device may In this case, one computer is called a client. Another computer can be considered a server, and each computer can be a part of the same computer system. The client and the server may each be part of a number of systems, servers, The present invention may include a system or component.

[0084] Any of the embodiments of the present disclosure may be implemented using hardware (e.g., application specific integrated circuits or Field Programmable Gate Arrays (FPGAs) and / or modular or using computer software with an integrated general purpose programmable processor. It should be understood that the term may be implemented in the form of control logic using the same techniques as those described herein. When used in a multi-core processor, the processor may include a multi-core processor on the same integrated chip or a single Multiple processors on the same circuit board or multiple processors networked together Based on the disclosure and teachings provided herein, one of ordinary skill in the art can easily design and implement the hardware and A combination of hardware and software may be used to implement embodiments of the present disclosure. The individual will be aware of and understand other ways and / or methods for

[0085] Any of the software components or functions described in this application may be implemented using, for example, Java (registered trademark) using any suitable computer language, such as, for example, The software executed by the processor may be implemented using conventional or object-oriented techniques. The software code may be implemented as stored and / or may be stored as a series of instructions or commands on a computer-readable medium for transmission. Suitable media include random access memory (RAM), read-only memory (ROM), and M), magnetic media such as hard drives or floppy disks, or compact disks Optical media such as disks (CDs) or DVDs (digital versatile disks), flash The computer-readable medium includes any such storage or transmission device. It may be a combination of the above.

[0086] Such programs may also be used to communicate over wired networks, including the Internet, in accordance with a variety of protocols. , using a carrier signal adapted for transmission over optical and / or wireless networks. Thus, the present invention provides a computer-readable medium according to an embodiment of the present invention. can be produced using such a programmatically encoded data signal. A computer readable medium encoded with the program code is packaged in a compatible device. The software may be digitized or made available on other devices (e.g., via internet download). Any such computer readable medium may be provided separately from the computer. on computer products (such as hard drives, CDs or entire computer systems), and may exist within a single computer, on different computer products within a system or network. The computer system may be present in or on the computer system itself. A monitor, printer or other suitable display device may be used to provide the user with It may also include rays. EXAMPLES

[0087] The following examples are provided to better illustrate the claimed invention and to limit the scope of the invention. All specific compositions, materials and methods described below are intended to be non-limiting. These specific compositions, materials and methods are within the scope of the present invention, in whole or in part. The following are not intended to limit the invention, but merely to illustrate specific embodiments within the scope of the invention. A person skilled in the art can easily make the same invention without exerting his creative ability and without departing from the scope of the present invention. Compositions, materials and methods such as those described herein may be developed while remaining within the scope of the present invention. It should be understood that many variations can be made in the techniques described herein. It is the intention of the inventors that such modifications are included within the scope of the present invention.

[0088] [Example 1]

[0089] This example demonstrates mouse cross-cancer genetics for cancer immunotherapy and tumor microenvironment research. 1 illustrates the identification of a child expression panel.

[0090] material and method

[0091] Public and private reference genome databases for non-tumor-bearing and tumor-bearing mouse models Marker genes were identified by combining the data. Oligonucleotide-based hybridization similar to endothelial sequencing The extraction / capture technologies used were Illumina Novaseq and BGI MGIseq. The results were further analyzed in mouse immune and tumor cell lines. Both were validated using a PCR assay and a panel of eight controls.

[0092] result

[0093] Based on the immune signature analysis, the inventors identified the tumor interface tumor microenvironment and immune response Surface markers and transcriptome biomarkers for the immune system, a key pathway for 1080 genes, including markers, as well as internal reference genes for data normalization (Table 1 The validation panel consisted of a set of well-characterized gene expression profiles in each cell type. The present inventors also demonstrated good consistency in the gene panel (see Figures 2 and 3). Based on expression, the cell fractions in the validation panel were estimated (see FIG. 4).

[0094] Use our Mouse Cancer Immunotherapy NGS Panel to streamline and accelerate your preclinical research. This allows researchers to characterize tumor-immune interactions in a rapid and cost-effective manner, which could improve response rates. With high content data, we have the ability to better understand the response versus the lack of Provides enhanced insight into the tumor microenvironment following treatment with immunotherapy, which may be an immunotherapy companion This ultimately resulted in the disease being effectively transferred to the clinic for earlier identification of the diagnosis.

[0095] This disclosure refers to certain embodiments, some of which are preferred embodiments. Although specifically shown and described herein, it is understood that those skilled in the art will appreciate that the present disclosure is not to be construed as being limited to the spirit and scope of the present disclosure. It should be understood that various changes in form and detail may be made without departing from the spirit and scope of the invention.

[0096] [Table 1(1)] [Table 1(2)] [Table 1(3)]

Table 1(4)

Table 1(5)

Table 1(6)

Table 1(7)

Table 1(8)

Table 1(9)

Table 1(10)

Table 1(11)

Table 1(12)

Table 1(13)

Table 1(14)

Claims

1. 1. A method for assessing an immune response in a mouse model, comprising: Obtaining a sample from the mouse model; detecting the expression level of a panel of genes in a sample, said panel comprising:

1. It comprises at least 50 genes selected from the group shown in comparing the expression levels of said panel to a reference expression level; and Evaluating immune responses in mouse models A method comprising:

2. The method of claim 1 , wherein the mouse model has a tumor.

3. The method of claim 1 , wherein the sample is a tumor tissue.

4. The method of claim 1 , wherein the mouse model is treated with a therapy.

5. The method of claim 4, wherein the therapy is immunotherapy.

6. 10. The method of claim 1, wherein the expression of the panel of genes is detected using NGS.

7. The immune response is assessed by determining the amount or activation of immune cells in the sample. wherein the immune cells are B cells, dendritic cells, macrophages, monocytes, natural killer cells, 2. The method according to claim 1, wherein the T cell is selected from the group consisting of CD4+ T cells, CD8+ T cells and CD8+ T cells. Method of posting.

8. administering a treatment to the mouse model; Obtaining a sample from the mouse model; detecting the expression level of a panel of genes in a sample, said panel comprising:

1. It comprises at least 50 genes selected from the group shown in comparing the expression levels of said panel to a reference expression level; and Evaluating immune responses in mouse models A method for evaluating a treatment in a mouse model comprising:

9. Primers for detecting expression of a panel of genes in samples from mouse models 1. A kit for assessing an immune response in a mouse model, comprising:

2. A kit comprising at least 50 genes selected from the group shown in Table 1.

10. A probe for enriching mRNA for a panel of genes in samples from mouse models A kit for assessing immune responses in a mouse model comprising a panel comprising at least 50 genes selected from the group shown in Table 1.

11. A protocol for detecting the expression levels of a panel of genes in samples derived from a mouse model. A microarray for evaluating immune responses in a mouse model, comprising: The panel comprises at least 50 genes selected from the group shown in Table 1. array.

12. A non-transitory computer readable medium having instructions stored thereon, the instructions being transmitted to a processor. When executed by the processor, the instructions cause the processor to: Detecting the expression level of a panel of genes in a sample, said panel comprising: a search comprising at least 50 genes selected from the group comparing the expression levels of said panel with a reference expression level; and Assessment of immune responses in mouse models A computer-readable medium for causing a

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